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WO1998050538A1 - LIGNEES CELLULAIRES D'ENCAPSIDATION RETROVIRALES ENDOGENES DE $i(MUS DUNNI) - Google Patents

LIGNEES CELLULAIRES D'ENCAPSIDATION RETROVIRALES ENDOGENES DE $i(MUS DUNNI) Download PDF

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WO1998050538A1
WO1998050538A1 PCT/US1998/009452 US9809452W WO9850538A1 WO 1998050538 A1 WO1998050538 A1 WO 1998050538A1 US 9809452 W US9809452 W US 9809452W WO 9850538 A1 WO9850538 A1 WO 9850538A1
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cells
virus
vector
leu
retroviral
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PCT/US1998/009452
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A. Dusty Miller
Greg Wolgamot
Lynn Bonham
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Fred Hutchinson Cancer Research Center
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Priority to AU75672/98A priority Critical patent/AU7567298A/en
Publication of WO1998050538A1 publication Critical patent/WO1998050538A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/10022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/10051—Methods of production or purification of viral material
    • C12N2740/10052—Methods of production or purification of viral material relating to complementing cells and packaging systems for producing virus or viral particles

Definitions

  • Retroviral vectors promote the transfer of genes into a variety of cell types from many animal species. Retroviral vectors are among the primary vehicles used for gene transfer into human somatic cells because of their ability to transfer genes efficiently into cells that are difficult to transfect by other methods.
  • a critical element in the production of the components to carry out retroviral mediated gene transfer is the cell that generates the retroviral particles carrying the gene to be transferred. These cells are called “packaging cells” because they "package" the retroviral vector which carries the gene of interest into a delivery vehicle, the retroviral particles.
  • Packaging cell lines are designed to synthesize all retroviral proteins required for assembly of high-titer infectious virus, but should not produce any replication-competent virus.
  • the retroviral vector consists of DNA sequences intended for transfer flanked by signals present at the ends of the retroviral genome, and the packaging cells are designed to produce all of the retroviral proteins and promote "packaging" of the retroviral RNA into virions. Retroviral vectors produced by using packaging cells can thus infect cells but cannot replicate further.
  • Retrovirus packaging cells provide useful tools for a variety of gene transfer applications. However, not all cell types can be efficiently infected by using the available packaging cell lines.
  • the range of cells that are infectable by a retroviral particle is primarily determined by the envelope proteins of the virus and the presence of appropriate receptors for this protein on the surface of target cells. For example, viruses that infect human cells can be separated into eight groups based on the use of different receptors for cell entry.
  • Recent improvements include the design of packaging cells to produce vectors having a vesicular stomatitis virus G protein coat for expanded host range (Burns et al . , Proc .
  • Packaging cells which produce amphotropic retrovirus were developed over ten years ago and are still commonly used because of the wide range of cell types from different species, including humans, that these vectors can transduce.
  • packaging cells have been developed based on gibbon ape leukemia virus (GALV) (Miller et al . , J. Virol. 65:2220-2224 (1991)) that produce vectors that use a different receptor than the prior known amphotrophic retroviruses for cell entry, and are capable of transducing myeloid, lymphoid, and airway epithelial cells at higher rates than amphotropic vectors do (Bauer et al . , Blood 86:2379-2387 (1995); Bayle et al . , Hum. Gene Ther. 4:161-170; Bunnell et al . , Proc. Natl. Acad. Sci. USA 92:7739-7743 (1995); von Kalle et al . , Blood 84:2890- 2897) .
  • GALV gibbon ape leukemia virus
  • the GALV and amphotropic retrovirus receptors are related phosphate transport proteins that exhibit wide, but different, patterns of tissue specific expression (Kavanaugh et al., Proc. Natl. Acad. Sci. USA 91:7071-7075 (1994)).
  • the GALV receptor Glvr-1 (Pit-1) is most highly expressed in bone marrow, while the amphotropic receptor Ram-1 (Pit-2) is most highly expressed in the heart. It has been shown that 10A1 murine leukemia virus can use either mouse or human Glvr-1 or rat or human Ram-1 for cell entry (Miller et al . , J. Virol. 68:8270-8276 (1994) ) .
  • the invention provides a cultured packaging cell for producing a replication-defective retroviral vector particle.
  • the packaging cell is a vertebrate cell capable of expressing and assembling retroviral proteins, and comprises a vector encoding a retroviral envelope protein having amino acid residues of the Mus dunni endogenous virus (MDEV) or fragment thereof, that direct binding of the retroviral particle to the MDEV retroviral receptors on a target cell.
  • MDEV Mus dunni endogenous virus
  • the packaging cell further comprises a vector encoding retroviral Gag and Pol proteins, such that upon expression of the vectors in the presence of a vector having a sequence of a heterologous gene of interest, a replication-defective retroviral vector particle is produced that binds to the MDEV retroviral receptors of target cells.
  • the retrovirus gag and pol genes can be from, for example, Moloney murine leukemia virus.
  • the cultured packaging cell can be an avian or mammalian cell capable of expressing and assembling retroviral proteins.
  • the vectors encoding the retroviral Env protein, the retroviral Gag and Pol proteins, and the vector comprising a heterologous gene of interest can be integrated in a chromosome of the packaging cell.
  • the invention provides a method for producing a replication-defective retroviral vector particle comprising a heterologous gene of interest.
  • the method comprises transducing or transfecting a retroviral packaging cell with (a) a replication defective virus particle which comprises virus RNA transcribed from a recombinant DNA provirus, the provirus comprising virus long terminal repeat sequences (LTRs) , a retrovirus packaging sequence, and a heterologous gene, or (b) a vector comprising the provirus.
  • a replication defective virus particle which comprises virus RNA transcribed from a recombinant DNA provirus, the provirus comprising virus long terminal repeat sequences (LTRs) , a retrovirus packaging sequence, and a heterologous gene, or (b) a vector comprising the provirus.
  • LTRs virus long terminal repeat sequences
  • the packaging cell can be a vertebrate cell capable of expressing and assembling retroviral proteins and having (i) an integrated vector encoding a retroviral envelope protein having amino acid residues of the Mus dunni endogenous virus (MDEV) that direct binding of the retroviral particle to the MDEV retroviral receptors on a target cell, and (ii) an integrated vector encoding retroviral Gag and Pol proteins.
  • the sequences which encode the retroviral Env protein, the retroviral Gag and Pol proteins, and the vector comprising the heterologous gene of interest are expressed, producing a replication-defective retroviral vector particle that binds to MDEV retroviral receptors of target cells.
  • methods for transiently producing a replication- defective retroviral vector particle comprising a heterologous gene of interest .
  • the method comprises transforming a transfecting vertebrate cell capable of expressing and assembling retroviral proteins with (i) a first vector comprising a retroviral gag gene; (ii) a second vector comprising a retroviral pol gene; (iii) a third vector comprising a Mus dunni endogenous virus env gene or a fragment thereof; and (iv) a (a) replication-defective virus particle which comprises virus RNA transcribed from a recombinant DNA provirus, the provirus comprising a virus long terminal repeat sequences (LTRs) , a retrovirus packaging sequence, and a heterologous gene, or (b) a vector comprising the provirus.
  • LTRs virus long terminal repeat sequences
  • the transfected or transformed cell is capable of transiently expressing the protein encoded by the M. dunni endogenous virus env gene, the retrovirus gag and pol genes, and the product of the heterologous gene of interest.
  • a replication-defective retroviral vector particle is transiently produced which combined to Mus dunni endogenous virus receptor of target cells.
  • the retrovirus pol gene can be combined on a single vector.
  • the invention provides a replication-defective retroviral vector particle containing the heterologous gene of interest produced by these methods.
  • the present invention provides retrovirus packaging cell lines based on the Mus dunni endogenous virus (MDEV) class of retrovirus.
  • MDEV packaging cells produce vectors capable of using a MDEV receptor for cell entry.
  • the Mus dunni endogenous virus is capable of infecting a variety of cell types across a wide host range including mouse, rat, hamster, quail, cat, dog, baboon and human.
  • the MDEV receptor is present on a variety of cells rendering MDEV pseudotype packaging cells useful in methods of mammalian and particularly human gene transfer.
  • the MDEV packaging cells are a stable and reproducible source of retroviral particles. Clones may be isolated from these populations that produce high titer virus.
  • the packaging cell lines of the invention can be selected and cloned for other desirable properties, such as stability of in vivo growth, lack of production of helper virus, lack of reinfection by viral particles packaged in the cell, stability from genetic rearrangement and recombinational events, resistance to complement lysis, and improved ability to infect cells from higher mammals.
  • the replication defective virus vectors produced by the MDEV packaging cell lines of the present invention permit the transfer of a wide variety of heterologous nucleic acid segments.
  • the packaging cell line is transduced or transfected with a replication defective virus vector, or a DNA construct having a strand corresponding to or complementary to a replication defective viral vector, containing the heterologous gene(s) of interest.
  • Representative genes useful in the present invention include, among others, those which encode, for example, blood clotting factors, adenosine deaminase, interleukins, interferons, GM-CSF, G-CSF, erythropoietin and other cytokines, receptors, cystic fibrosis transmembrane conductance regulator (CFTR) , tumor suppressors, antisense RNAs, and vaccine antigens.
  • CFTR cystic fibrosis transmembrane conductance regulator
  • the vector comprises a sequence capable of providing retroviral long terminal repeats (LTRs) , a sequence required for reverse transcription, a retroviral packaging sequence, and the gene sequence of interest.
  • a DNA construct includes the LTRs necessary for host cell genome incorporation and expression. Following synthesis the viral DNA is integrated into cellular DNA so that the ends of the LTRs are directly joined to cellular sequences to form a stable structure (e.g., the provirus) .
  • the vector component necessary for reverse transcription does not necessarily include sequence coding for reverse transcriptase, but rather, includes a replication initiation site and a polypurine tract.
  • a packaging signal preferably specific for the retroviral vector of interest, is included in the vector.
  • Donor and acceptor splice sites and internal ribosome entry sites may also be present in the replication defective virus vectors.
  • the splice sites enable the expression of additional heterologous inserted nucleic acid sequences.
  • the replication defective virus vector need not include one or more of the sequences corresponding to the viral genes gag, coding for the viral core proteins, pol , coding for the viral RNA-dependent DNA polymerase (reverse transcriptase and other enzymatic proteins) , or env, coding for the viral envelope proteins.
  • the sequences can be omitted or rendered defective by mutagenesis.
  • the gene of interest may be linked to a heterologous promoter.
  • a suitable promoter is well within the level of ordinary skill in the art.
  • a wide variety of promoters have been described in the literature including both viral and cellular promoters.
  • Viral promoters include the immediate early cytomegalovirus promoter (Boshart et al . , Cell 41:521-530, 1985), the murine leukemia virus (MLV) LTR promoter (Weiss et al . , RNA Tumor Viruses, Cold
  • Cellular promoters include but are not limited to the mouse metallothionien-1 promoter (Palmiter et al . , U.S. Patent No. 4,579,821). Alternative splicing may also be exploited to facilitate the expression of polycistronic genes in the vector.
  • one of the proteins encoded by the vector is translated from the full length vector RNA while partial splicing from a splice donor site near the 5' LTR to a splice acceptor just upstream of a downstream gene yields a transcript that encodes the downstream gene product. Because vector replication involves an RNA intermediate, construction of the vector containing inserted gene(s) should permit full-length transcription of vector genome.
  • the replication defective retrovirus vector particles packaged by the MDEV cell lines in accordance with the present invention will often contain at least one exogenous (heterologous) gene.
  • Heterologous genes encompass DNA (or RNA corresponding to the DNA) encoding proteins or peptides of interest and RNA molecules such as antisense RNA.
  • DNA sequences encoding proteins of interest include genes, cDNAs and minigenes .
  • a retroviral vector will contain the heterologous gene or other DNA which is desired to be transferred to the cells of the intended recipient .
  • Heterologous genes can code for the replacement or substitute of a defective or missing enzyme or other protein, RNA molecule or ribozyme in the patient, or encode therapeutic proteins or RNA molecules normally not present in the patient.
  • the enzyme or other protein may function within a cell, or may be secreted and circulate in the body, such as hormones and blood factors .
  • Genes which code for proteins whose levels do not have to be precisely controlled, and/or genes which cause disease by virtue of a single defect, are particularly suitable for insertion in a retroviral vector packaged by a MDEV packaging cell line of the present invention.
  • Selectable markers can also be included in the replication defective retroviral vectors packaged according to the present invention, for investigative or experimental purposes, or to provide a means to select for cells containing the replication defective retroviral vectors. These markers include the neomycin and hygromycin phosphotransferase genes that confer resistance to G418 and hygromycin, respectively.
  • markers include the mutant mouse dihydrofolate reductase gene (dhfr*) which confers resistance to methotrexate, the bacterial gpt gene which allows cells to grow in medium containing mycophenolic acid, xanthine, and aminopterin, the bacterial hisD gene which allows cells to grow in medium without histidine but containing histidinol, and the multidrug resistant gene (mdr) which confers resistance to a variety of drugs. These markers are dominant selectable markers and allow chemical selection of most cells expressing these genes. Suicide genes may also be contained within the vectors packaged by the MDEV cell lines of the present invention. Such genes provide a means to selectively kill cells containing the retroviral RNA. For example, the tk gene (Culver et al . , Science 256:1550-1552 (1992)) may be used in combination with gancyclovir to selectively kill transduced cells .
  • the tk gene Culver et al . , Science
  • the exogenous gene for insertion in the vector can be an intronless cDNA copy of an mRNA encoding a gene product of interest.
  • Large inserts can be placed in the replication defective retroviral vectors, but generally the gene(s) of interest and any attendant regulatory sequences should be no more than up to approximately 8 to 11 kb in size.
  • the 5' and 3 ' noncoding regions of the cDNA can be trimmed to reduce the size of the insert and to remove potential polyadenylation signals that may occur in the 3' end of cDNAs . It may be preferable to insert the cDNA in the same transcriptional orientation as the viral LTR.
  • Antisense RNAs can be expressed by reversing the orientation of the cDNA with respect to a promoter.
  • inserts include intronless "minigenes” which include normal genes from which the introns have been removed and those with gagated coding regions . Entire genes containing introns can also be inserted, and typically will be inserted in reverse orientation to prevent removal of the introns during vector replication.
  • Packaging cell lines of the present invention may be constructed and optimized using a variety of strategies.
  • such strategies are designed to reduce the chance of recombination between a helper construct (i.e., one or more constructs that provide trans-acting proteins required for production of replication defective retroviral vector particles) and the vector that may result in the production of helper virus.
  • helper virus means undesirable replication-competent retrovirus produced from the integrated proviral genome in some packaging cells by genetic recombination and repair of the defective retroviral vector proviral genome.
  • helper constructs in which the packaging signal (s) have been deleted helper constructs in which the gag, pol and env genes are split into two or more separate transcriptional units, e.g., containing gag-pol and env, or helper constructs in which the gag-pol and env genes are split into two separate transcriptional units and which contain mutations (e.g., by insertions of linkers) and deletions in the gag-pol and env transcriptional units.
  • the 3' LTRs in separate transcriptional units can be replaced with polyadenylation signals from SV40, thereby requiring an additional recombinational event to generate helper virus.
  • helper constructs are cotransfected with the vector of the present invention thus providing the required trans-acting proteins and allowing virus particle production.
  • Trans-acting proteins may also be provided by packaging cell lines that are designed to provide all viral proteins but not to package or transmit the RNAs encoding these functions. These packaging cell lines contain the replication defective retroviral vector genome of interest, and expression of the trans-acting proteins permits the production of packaged retroviral vectors.
  • the trans-acting viral proteins may be provided in a transient or inducible manner.
  • Trans-acting viral genes may be placed under the control of an inducible promoter such as the tetracycline-responsive promoter (Gossen and Bujard, Proc. Natl. Acad. Sci. USA 89:5547-5551, 1992 and Pescini et al . , Biochem. Biophys. Res. Comm. 202:1664-1667, 1994) .
  • packaging may be indicated by inducing the promoter.
  • Cells suitable for use in preparing packaging cell lines of the present invention are derived from vertebrates and include, for example, avian, primate, porcine, human, murine, canine etc. Particularly preferred cells for preparing packaging cells are NIH 3T3, however, other suitable cells are readily available.
  • the MDEV packaging cells are produced by introducing DNA constructs which direct the expression of trans-acting Gag, Pol and MDEV envelope proteins that are required for packaging replication defective retroviral particles.
  • Methods for introducing such constructs include, for example, calcium phosphate precipitation (Wigler et al . , Cell 14:725 (1978); Corsaro and Pearson, Somatic Cell Genetics 7:603 (1981); Graham and Van der Eb, Virology 52:456 (1973); lipofection (Feigner et al . , Proc. Natl. Acad. Sci. USA 84:7413-7417 (1987)), microinjection and electroporation (Neumann et al . , EMBO J.
  • the cells can also be transduced with virus, such as SV40, CMV and the like.
  • virus such as SV40, CMV and the like.
  • cloned DNA molecules may be introduced by infection of susceptible cells with viral particles.
  • the gag and pol genes may be derived from a wide variety of retroviruses, and within a preferred embodiment the gag and pol genes are derived from Moloney murine leukemia virus (MoMLV) .
  • MoMLV Moloney murine leukemia virus
  • methods for transiently producing replication-defective virus particles by transducing or transfecting a vertebrate cell with a first vector comprising a retrovirus gag gene, a second vector comprising a retrovirus pol gene, a third vector comprising a MDEV env gene, or a fragment thereof encoding a receptor binding fragment; and a replication defective virus particle which comprises virus RNA transcribed from a recombinant DNA provirus, the provirus comprising virus long terminal repeat sequences (LTRs) , a retrovirus packaging sequence, and a heterologous gene, or a vector comprising said provirus, wherein the cell is capable of expressing and assembling retroviral proteins.
  • LTRs virus long terminal repeat sequences
  • the transfected or transformed cell transiently expresses the proteins encoded by the M. dunni endogenous virus env gene, or a fragment of the Env protein capable of binding to the M. dunni endogenous receptor on a target cell, and also expresses the retrovirus Gag and Pol proteins as well as the product encoded by the heterologous gene.
  • a replication-defective retroviral vector particle is produced which can bind to M. dunni endogenous virus receptors of target cells.
  • Transient packaging can also be accomplished using a first vector which contains both the retrovirus gag and pol genes in place of the first and second vectors described above. (See, for example, Burns et al . , Proc. Natl. Acad.
  • replication defective virus particles produced transiently or by the packaging cell lines of the present invention all are capable of binding to MDEV receptors on target cells and provide means for the transfer of a wide variety of heterologous nucleic acid segments.
  • a packaging cell of the present invention provides replication defective retroviral particles capable of transducing cells (i.e., an infectious virus having a ribonucleoprotein core particle surrounded by a membrane containing MDEV envelope protein, or fragments thereof which are capable of binding to the MDEV receptor on target cells) containing the vectors as described herein.
  • the production of undesirable helper virus can be detected in a variety of ways, including, e.g., vector rescue assays in which cells containing but not producing a selectable replication-defective viral vector are transduced with the test virus and assayed for production of the vector.
  • Rescue of the vector can be detected by passaging the cells to allow virus spread and assaying medium exposed to these cells for the selectable viral vector in a standard colony assay.
  • Representative assays include the S + L " assay described by Bassin et al . (Nature 229:5646, (1971), incorporated herein by reference) and marker rescue described by Miller et al . (Meth. Enzymol . 217:581-599 (1993), incorporated herein by reference) .
  • the replication defective retroviral vectors packaged by the MDEV packaging cell lines of the present invention provide the means for gene transfer in a wide range of animals species, including, e.g., experimental and domestic animals, livestock (e.g., sheep, cows, horses), birds (e.g., chickens), cats, rats, mice, hamsters, dogs, monkeys and primates (e.g., chimpanzees, macaques, and monkeys, and humans) .
  • livestock e.g., sheep, cows, horses
  • birds e.g., chickens
  • cats e.g., rats, mice, hamsters, dogs, monkeys and primates
  • primates e.g., chimpanzees, macaques, and monkeys, and humans
  • the particles produced according to the invention are useful for infecting cells in preimplantation embryos, which embryos when implanted in an animal creates a transgenic animal or an animal which expresses a gene product that it would normally not produce.
  • the replication defective retroviral particles packaged in a MDEV packaging cell line of the invention are used to infect (transduce) target cells, such as, for example, those which are defective in expression of the gene of interest, or which can act to secrete the desired protein.
  • transduction is meant the process by which non-viral genes are transferred and expressed in a host cell by a viral vector. Transduction can take place ex vivo or j_n vivo.
  • the targeted cells e.g., lymphocytes, bone marrow, hematopoietic cells and hematopoietic stem cells, fibroblasts, hepatocytes, endothelial cells, benign or malignant tumor cells, etc.
  • the targeted cells e.g., lymphocytes, bone marrow, hematopoietic cells and hematopoietic stem cells, fibroblasts, hepatocytes, endothelial cells, benign or malignant tumor cells, etc.
  • the cells are infected by the replication defective virus particles containing the gene of interest, and the cells are returned (or transplanted) to the host.
  • typically medium containing the recombinant replication defective virus particles is incubated with the target cells.
  • the target cells may be cultivated ex vivo to expand their numbers in primary cell cultures. Transduction is typically during the early days of host cell culture, and may be accomplished by co-cultivating the target cells with a cell line producing replication defective virus vectors.
  • the target cells are not necessarily cultured prior to transduction and replacement in the host patient.
  • the replication defective virus particles can be administered to the host in a wide variety of ways.
  • the particular mode of administration will depend upon several factors, including, among others, the particular use intended, the host being treated, the tissue targeted for transduction, the gene product of interest, the general health of the patient, etc., but will generally be administered intradermally, subcutaneously, intramuscularly, topically (e.g., aerosol, such as via a nebulizer), intravenously, intraperitoneally, or the like.
  • the vectors may be administered to tissues and organs (e.g., via a catheter) such as the lungs, bladder, urethra, uterus, liver, heart and circulatory system, kidney, bone marrow, brain, lymphoid tissues, stomach, small intestine, large intestine, colon and prostate.
  • the dosages of the replication defective virus vectors produced according to the invention will be determined through empirical experiments such as dose escalation studies and the like. It must be kept in mind that the materials of the present invention may be employed in serious disease states, that is, life-threatening or potentially life threatening situations. In such cases, in view of the ability of the replication defective virus particles produced by the present invention to infect a wide variety of vertebrate cells, it is possible and may be felt desirable by the treating physician to administer substantial excesses of these viral particles.
  • Nomenclature cells that contain a retroviral vector and/or contain and express a retrovirus are indicated by the cell name followed by a slash and the name of the vector, e.g., dunni/LN are Mus dunni cells that contain the LN retroviral vector and G355/LAPSN are G355 cat cells containing the retroviral vector LAPSN.
  • dunni/LN Mus dunni cells that contain the LN retroviral vector
  • G355/LAPSN are G355 cat cells containing the retroviral vector LAPSN.
  • a retroviral vector in its viral form is indicated by the vector name followed, in parentheses, by the name of the helper virus or packaging cells used to pseudotype the vector, e.g., LAPSN(PT67) refers to the viral form of the LAPSN retroviral vector packaged by PT67 cells, which express the 10A1 murine leukemia virus envelope and LAPSN (PA317) refers to the viral form of the LAPSN retroviral vector packaged by PA317 cells, which express the amphotropic MLV envelope.
  • the pseudotype of a retroviral vector refers only to the viral envelope protein present on the vector virions that determines the cell-surface receptor utilization pattern of the vector, irrespective of the origin of the Gag and Pol proteins.
  • D17 dog cells ATCC CCL 183
  • LGPS cells Miller et al . , J. Virol. 65:2220-2224 (1991)
  • packaging cell lines PA317 Miller and Buttimore, Mol . Cell . Biol. 6:2895-2902 (1986) and U.S. Patent No. 4,861,719)
  • PG13 Massiller et al . , J. Virol. (1991) and U.S. Patent No. 5,470,726)
  • PT67 Miller and Chen, J. Virol. 70:5564- 5571(1996)
  • PM571 ((Miller and Miller, J. Virol.
  • FLYRD Cosset et al . , J. Virol. 69:7430-7436 (1995)
  • PE501 Mass et al . , BioTechniques 7:980-990 (1989) were grown in Dulbecco's modified Eagle's medium with 10% fetal bovine serum (FBS) .
  • FBS Dulbecco's modified Eagle's medium with 10% fetal bovine serum
  • G355 feline embryonic glial cells (Dunn et al., J. Virol. 67:4704-4711 (1993)) were grown in McCoy's modified Eagle's medium supplemented with 15% FBS.
  • CHO cells were grown in cMEM with 5% FBS.
  • This Example describes the molecular cloning of M. dunni endogenous virus (MDEV) and relationship by nucleic acid hybridization to other retroviruses .
  • MDEV M. dunni endogenous virus
  • a strategy was initially employed that involved use of radiolabeled DNA from a variety of cloned retroviruses as potential probes for the detection of plasmids containing MDEV sequences.
  • MDEV involved construction of a plasmid library from unintegrated viral DNA and screening of the library for clones containing MDEV sequences by using a probe derived from viral RNA.
  • Extrachromosomal DNA was harvested from M. dunni cells 24 hours after infection with a mixture of MDEV and
  • LAPSN virus On day one, M. dunni cells were seeded at 2 x 10 6 cells per 14 cm-diameter dish in 60 dishes. On day two, the cells were infected with MDEV plus LAPSN virus produced from GL8cl6 cells (at a multiplicity of infection (MOI) of 2.5 based on the LAPSN vector) with 4 ⁇ g/ml Polybrene . Twenty- four hours after infection, extrachromosomal DNA was isolated by the method of Hirt (Hirt, B., J. Mol. Biol. 26:365-369 (1967); incorporated herein by reference in its entirety) .
  • Hirt Hirt, B., J. Mol. Biol. 26:365-369 (1967); incorporated herein by reference in its entirety
  • RNA extracted from virus produced from G355 cat cells infected with MDEV and LAPSN (GL8cl6) .
  • the viral RNA was isolated from virus produced from cat cells, rather than from M. dunni cells, to minimize the presence of sequences reactive with M. dunni DNA sequences that were expected to be present in the viral DNA library.
  • GL8cl6 cells were seeded at lxlO 6 cells/10 cm plate.
  • pellets were resuspended in a total of approximately 500 ⁇ l ice cold TNE buffer (10 mM Tris, pH 7.5, 100 mM NaCl, 1 mM EDTA) .
  • Viral RNA was extracted from the pellets as described (MacKenzie et al . , J. Virol. 68:6924-6932 (1994); incorporated herein by reference in its entirety) and precipitated at 70°C, overnight. RNA was pelleted in a microcentrifuge (EPPENDORF) at 4°C for 30 minutes, and the pellet was resuspended in 1 ml distilled water. Polyadenylated RNA was selected using a cellulose affinity column (Mini-Oligo (dT) Cellulose Spin Column Kit (5' -> 3', Boulder, CO)) according to the manufacturer's instructions.
  • a cellulose affinity column Mini-Oligo (dT) Cellulose Spin Column Kit (5' -> 3', Boulder, CO)
  • cDNA Complementary DNA
  • reaction conditions were similar to those described (Arya et al., Prep. Biochem. 10:483-493 (1980); incorporated herein by reference in its entirety) .
  • the reaction mix contained viral RNA in 10 ⁇ l distilled water, 50 mM Tris, pH 8.1, 10 mM DTT, 50 mM NaCl , 3 mM magnesium acetate, 0.6 mM magnesium chloride, 1 mM each of dGTP, dATP, and dTTP, 100 ⁇ Ci c-- 32 P-dCTP (800 Ci/mmol) , 500 ⁇ g/ml oligo dT primers, 0.5 units/ ⁇ l Promega RNasin, and 70 units of MoMLV reverse transcriptase (Stratagene) .
  • the reaction was incubated at 37°C for one hour and cDNAs were separated from free nucleotides using a size exclusion column (Sephadex G-50) . This probe was then tested by Northern blot analysis for specific hybridization to MDEV transcripts and not to RNA from uninfected cells or to RNA from cells infected with amphotropic virus.
  • the Southern blot was analyzed to determine which enzymes would be useful in cloning the Mus dunni endogenous virus (MDEV) DNA. Eco RI appeared to cut only once within MDEV. Based on the results, the supercoiled DNA was digested with Eco RI and cloned into the BLUESCRIPT plasmid (Stratagene), generating a library with a complexity of 10 5 . The library was screened using standard procedures, and positive clones were confirmed by Southern blot analysis (Maniatis et al . , Molecular Cloning: a Laboratory Manual, CSH Laboratory Press: Cold Spring Harbor, N.Y. (1989)).
  • the MDEV plus LAPSN cDNA probe identified multiple clones of LAPSN and clones of a retrovirus presumed to be MDEV, containing both one and two LTRs, from the library. No positive clones other than LAPSN and the presumptive MDEV were identified. One positive clone, pMDEV9 , was selected for further sequence analysis .
  • Regions of poor sequence were resequenced after making appropriate primers and using the ABI PRISM dye terminator kits. Both strands of MDEV were completely sequenced, and the sequences were assembled into contigs and further analyzed using SEQUENCHER 3.0 (Gene Codes Corporation, Inc., Ann Arbor, MI). The nucleotides of the MDEV genome were numbered beginning with the presumptive cap site, the first nucleotide of the R region. This putative cap site was identified by comparison of MDEV to the VL30 element VL3 (accession X03489) in which the cap site has been mapped (Rotman et al . , Nuc . Acids Res . 14:645-656 (1986)).
  • the complete nucleotide sequence of MDEV (SEQ ID NO. 1) and the deduced amino acid sequences of the glycosylated Gag (SEQ ID NO: 2) , Gag (SEQ ID NO: 3), Pol (SEQ ID NO: 4) and Env (SEQ ID NO: 5) of MDEV have been deposited into GenBank under accession number AF053745.
  • pMDEV9 The retroviral sequence in one of the presumptive MDEV clones (pMDEV9) was related to the virus which was activated from the M. dunni cells, cytoplasmic RNA samples isolated from both activated and unactivated M.
  • dunni-v a type of Mus dunni tail fibroblast that when grown to confluency, secretes molecules that render the culture medium viscous (Eiden et al . , J. Virol. 67:4056-4061 (1993) and Eiden et al . , J. Virol. 68:626-631 (1994)) and from uninfected and MDEV-infected G355 cells were analyzed by Northern blot, using the insert from pMDEV9 as a probe. The probe hybridized to 8.6 kb genomic and 3.7 kb subgenomic RNAs in both activated M.
  • dunni-v cells and G355 cells producing MDEV demonstrating the specificity of the probe for the MDEV transcripts present in infected cells.
  • the pMDEV9 probe did not hybridize to RNA from cells infected with AM-MLV plus LAPSN vector, indicating limited sequence similarity between MDEV and AM-MLV or LAPSN.
  • the probe did not hybridize to RNA from unactivated M. dunni cells transduced with LAPSN vector which do not produce MDEV or to uninfected G355 cells, indicating that MDEV is not normally transcribed in unactivated M. dunni cells or uninfected G355 cells. MDEV was not successfully activated from M. dunni-nv (a type of M.
  • dunni tail fibroblast that when grown to confluency does not secrete molecules that render the culture medium viscous.
  • MDEV can infect and replicate in M. dunni-v and many other cells without further chemical treatment, indicating that all of the genes required for virion production and virus replication are present, and that the viral promoter can remain active.
  • MDEV is endogenous to the M. dunni genome, and related sequences are present in the genomes of laboratory strains of mice. To determine whether MDEV was actually present in the germline of the M. dunni mouse and not an acquired contaminant of the M. dunni cell line, Southern analysis was performed using the viral DNA insert from pMDEV9 as a probe to examine genomic DNA from a M.
  • dunni cell line and from the spleens of two M. dunni mice The genomic DNA samples were digested with Eco RI , which cuts once within the MDEV provirus, to allow detection of MDEV, its copy number, and whether its integration site(s) were the same in the various samples.
  • Eco RI which cuts once within the MDEV provirus, to allow detection of MDEV, its copy number, and whether its integration site(s) were the same in the various samples.
  • Two strongly hybridizing bands were visible in DNA from the M. dunni cells and these same bands were present in DNA from both M. dunni mice, indicating that MDEV is present in the germline of the M. dunni mouse at the same integration site as in the cell line. Given that the entire MDEV provirus was used as a probe, these two bands probably represent the two halves of one copy of MDEV.
  • a less intense band was visible at approximately 23 kb in the DNA of both mice, and at still lower intensity in the DNA from the M. dunni
  • the number of copies of elements closely related to MDEV was determined by Southern blot analysis of Xho I-digested DNA. Digestion of DNA containing a provirus with Xho I sites would be expected to produce a fragment of approximately 9 kb because there is one Xho I site in each LTR of the MDEV clone. Therefore, using the entire MDEV provirus as a probe on this blot, a 9 kb band was observed that coincides with that expected from MDEV in the M. dunni cells. Xho I-digested M.
  • dunni DNA also produced DNA fragments of approximately 6 and 3 kb which hybridized less well to MDEV.
  • MDEV pol and env DNA fragments as probes on this blot, the band at 6 kb hybridized to MDEV pol.-related sequences and the band at 3 kb hybridized to MDEV env-related sequences, while the presumptive MDEV band at 9 kb hybridized to both probes.
  • the 3 and 6 kb bands may comprise a second, MDEV-related element with a Xho I site in each LTR and an additional, internal Xho I site. This element may also be responsible for the large, somewhat indistinct -23 kb band seen on the blot of Eco RI -digested DNA.
  • MDEV Mus dunni endogenous virus
  • Example II Six molecular clones of MDEV were isolated from a library of extrachromosomal DNA from G355 cat cells infected one day earlier with MDEV essentially as described in Example I and by Bonham et al . (J. Virol. 71:4663-4670 (1997), which is incorporated by reference herein in its entirety) .
  • a library of unintegrated viral DNA was produced by digesting the extrachromosomal circular DNA from MDEV-infected G355 cells with Eco RI , and cloning it into pBSII KS+ (Stratagene Cloning Systems, La Jolla, CA) .
  • the library was screened using standard procedures and positive clones were confirmed by Southern blot analysis (Maniatis et al . , Molecular Cloning: a Laboratory Manual , CSH Laboratory Press: Cold Spring Harbor, N.Y. (1989) ) . Sequence analysis of the clones demonstrated that the MDEV Eco RI site was in the pol gene. Different orientations of the virus, the presence of one versus two LTRs, and the presence of extraneous DNA fragments in the pBSII KS+ Eco RI cloning site demonstrated that the clones were independent. A vector-rescue assay was employed to determine whether the molecular clones were competent to provide gag. pol , and env gene products in trans to a vector.
  • Each of the six clones was digested with Eco RI , religated at low concentration to rejoin the pol gene, and transfected into G355/LAPSN cells, but none of the clones were competent to rescue the LAPSN vector (Miller et al . , Proc. Natl. Acad. Sci. USA 91:78-82, (1994)). However, three clones were competent to rescue LAPSN when cotransfected with pSX2 (Miller and Chen, J. Virol. 70:5564-5571 (1996)), a plasmid that expresses ' the 10A1 MLV envelope protein.
  • PCR polymerase chain reaction
  • Templates included genomic DNA from unactivated M. dunni cells and G355/LAPSN+MDEV cells, as well as negative controls including no template and DNA from uninfected G355 cells. Amplification reactions performed on DNA from uninfected G355 cells or performed with no DNA did not yield detectable product.
  • PCR products from both unactivated Mus dunni cell DNA and G355/LAPSN+MDEV DNA were then cloned into plasmid pT7Blue (Novagen) according to the manufacturer's protocol.
  • Several clones from separate PCR amplifications of various templates were sequenced. Sequencing demonstrated that the frame-shift was due to an extra A residue at position 6168 within the first variable region of env generating the +1 frame-shift.
  • the sequence revealed additional changes peripheral to the frame-shift that affect the protein sequence. As it was unknown whether these additional changes represented errors by Taq polymerase, the mutation was corrected in two ways by site-directed mutagenesis.
  • the env mutation was corrected by site-directed mutagenesis to delete the single extra base pair that resulted in the frame-shift.
  • the env region was subcloned as a 2.8 kb Bam Hl-Xho I fragment of pMDEV9 into Bam Hi-Sal I linearized pBSII KS+.
  • the resulting plasmid, pEA9 was used as a template for the site-directed mutagenesis to correct the frame-shift mutation using the QUICKCHANGE site-directed mutagenesis kit (Stratagene Cloning Systems) .
  • MDEVMUTF 5 ' CAG GGT CAG AAA GGA AAG CTG CAA CAA GAA TG 3 ' ;
  • TTC TGA CCC TG 3'; (SEQ ID NO. 9) were designed to create a plasmid containing the corrected env gene .
  • the primers and the pEA9 template were subjected to thermocycling (one cycle at 95°C for 30 seconds, 16 cycles (95°C for 30 seconds, 55°C for 1 minute, 68 °C for 24 minutes) ) .
  • the products were digested with Dpn I and transformed into E. coli .
  • Plasmid DNA prepared from selected transformants were subjected to sequence analysis to identify a plasmid containing the corrected env gene.
  • One clone containing the frame-shift correction which was confirmed by sequence analysis, designated pEA9corr, was digested with Bam HI, Xmn I and Ahd I to isolate the 2.17 kb Bam Hl-Xmn I env gene fragment.
  • the 2.17 kb Bam Hl-Xmn I fragment was then subcloned into the 3948 bp fragment of pSX2 (Miller and Chen, J. Virol. 70:5564-5571, 1996) that had been prepared by digestion with Bsa Bl and partial digestion with Bam HI to generate pMDEV9ex.
  • the plasmid, pMDEV9ex contained the corrected env gene in the correct orientation relative to the pSX2 backbone .
  • wild-type env gene sequences were obtained by PCR amplification of templates containing the wild-type MDEV sequences using primers that overlap the start and stop codons of the MDEV env.
  • the templates for amplification included the plasmid pEA9corr, which contains the same envelope sequence as pMDEV9ex, DNA from unactivated M. dunni cells, and DNA from G355/LAPSN + MDEV cells. DNA from uninfected G355 cells and no DNA were used as negative controls for the PCR amplification reactions.
  • the primers (MDEV Env 1 5' GCC CAC CGT GTG CCA CCA TGA AGA AAC CCA CGA AGA CAA C 3' ; (SEQ ID NO. 10) and MDEV Env 2: 5' GCG GTT AAC ATA GCT CTA ATC CTA GAG CGA G 3' ; (SEQ ID NO. 11) ) were designed to amplify a fragment encoding the wild-type MDEV env gene flanked on the 5' end by a near consensus sequence (Kozak, Proc. Natl. Acad. Sci. USA 92:2662-2666 (1995) ) to optimize translation; an upstream Dra III site, and flanked on the 3' end by a Hpa I site.
  • the primers and the template containing the wild-type env gene were amplified using Pwo polymerase (Boehringer Mannheim) at one cycle of 2 minutes at 94°C, 10 cycles (15 seconds at 94°C, 30 seconds at 62°C, 80 seconds at 72°C) , 20 cycles (15 seconds at 94°C, 30 seconds at 62°C, 80 seconds cumulatively increased by 20 seconds cycle at 72 °C) , 7 minutes at 72 °C.
  • the reactions were stored at 4°C.
  • the PCR products from unactivated M. dunni DNA or G355/LAPSN + MDEV DNA were ligated into Bsa BI-Dra III digested, blunt-ended pSX2.
  • This vector fragment contained the MoMLV LTR promoter truncated at the 5 ' end to a Sau3 Al site just upstream of the transcriptional enhancers in the LTR, the MoMLV splice donor, the 10A1 splice acceptor, the early polyadenylation signal from SV40 and plasmid sequences derived from the poison-minus plasmid pML-1 (Lusky et al . , Nature 293:79-81 (1981)). Plasmids containing envelope PCR products with wild-type MDEV env sequences in the correct orientation relative to the LTR were termed the pMEX series of plasmids. Twenty clones were selected for analysis.
  • Plasmids pMEX dunni and pMEX plas id contain an envelope amplified from DNA of unactivated M. dunni cells and an envelope amplified from plasmid DNA containing the same envelope as pMDEV9ex. Sequence analysis of pMEX dunni shows that the mutation in pMDEV9 was corrected.
  • the twenty "pMex" MDEV envelope expression constructs were either transfected into the LGPS clone 91-22 (U.S. Patent No. 5,470,726, incorporated herein by reference) that had been previously infected with the retroviral marker vector LAPSN (Miller et al . , Proc. Natl. Acad. Sci. USA 91:78-82 (1994)) (yielding LGPS/LAPSN) or co- transfected with pLGPS (which expresses the Moloney MLV Gag and Pol) into G355/LAPSN.
  • the LAPSN vector contains the neomycin resistance gene and a human placental alkaline phosphatase gene flanked by retroviral LTR's.
  • the LGPS/LAPSN and G355/LAPSN cells do not contain the env gene required to produce functional vectors.
  • an irrelevant plasmid was transfected into the cells. One day post-transfection the culture medium was changed. The culture medium from each transfection was harvested at two days post-transfection. The medium was centrifuged at 4,000 x g for five minutes and aliquoted and stored at -80°C until titration.
  • Titers of LAPSN vector stocks were, determined by plating D17 target cells on day 1 at 5xl0 4 cells per 35 mm (diameter) well of a 6 well plate. The medium was replaced with fresh medium containing 4 ⁇ g/ml of Polybrene (Sigma, St. Louis, MO) and a dilution of the medium from the transfected cells in a final volume of 2 ml on day 2. The cells were fixed with glutaraldehyde and stained for alkaline phosphatase (AP) expression as described (Field-Berry et al., Proc. Natl. Acad. Sci. USA 89:693-697 (1992); which is incorporated herein by reference in its entirety) on day 4.
  • AP alkaline phosphatase
  • pMEX clones were screened to identify those capable of expressing functional envelope protein. All five clones amplified from plasmid DNA were functional. Five out of six clones amplified from G355/LAPSN + MDEV DNA were functional, but only two out of nine clones amplified from M. dunni cell DNA were functional. Elements related to MDEV exist in the M. dunni genome and some of the nonfunctional pMEX plasmids may carry related but defective envelopes. The transfection experiments indicated that the various functional MDEV env expression plasmids function similarly. Table 1 provides a comparison of representative MDEV env expression plasmids, 10A1 env expression plasmid (pSX2) and an empty vector.
  • Plasmid LAPSN titer (AP + FFU/ml) PBSII KS+ ⁇ 5 pSX2 lxlO 5 pMDEV9ex 2xl0 3 pMEXP lasmid 2x10 3 pMEX dunni 2x10 3
  • the titers are expressed as the mean of the values obtained from duplicate transfections .
  • Plasmid pMEX dunn ⁇ which was constructed using Mus dunni DNA as a PCR template, gave among the best titers and was used in the construction of the MDEV packaging cells.
  • plasmids were introduced into cells of LGPS clone 91-22 that express MoMLV Gag-Pol proteins. On day 1, LGPS clone 91-22 cells were plated in three dishes at 5xl0 5 per 6 cm diameter dish.
  • the cells were co- transfected with 5 ⁇ g DNA mixture of pMEX dunn ⁇ and a hygromycin phosphotransferase ( pt) gene contained in the plasmid pSV2 ⁇ 130hyg (obtained from Paul Berg, Stanford University) by calcium phosphate precipitation (Miller et al . , BioTechniques 7:980-990 (1989)).
  • the ratio of selectable marker plasmid to env expression plasmid was 1:20.
  • the medium was replaced on day 3, and on day 4 the cells were split into medium containing 0.4 mg/ml of hygromycin. Non-transfected control cells were completely killed by the hygromycin by day 10, so the cells were released from selection.
  • Packaging cell clones were subjected to two screening assays to determine which clone possessed the best ability to package a retroviral vector (Table 2) .
  • a primary screen aliquots of each clone were infected by LAPSN (PE501) at a multiplicity of infection (MOI) of approximately 1. Entry of the LAPSN vector pseudotyped by PE501 cells was unaffected by expression of the MDEV Env or MoMLV Gag-Pol proteins.
  • the primary screen was initialized by plating aliquots of the clones at 5xl0 4 cells per 3.5 cm diameter well of a 6 well dish on day 1. On day 2, the medium was replaced with fresh medium containing 4 ⁇ g Polybrene per ml and 200 ⁇ l of LAPSN (PE501) .
  • the LAPSN (PE501) stocks had previously found to have a titer of 5xl0 5 AP + FFU/ml on NIH 3T3 TK " cells using the procedure as described by Fields-Berry et al., Proc. Natl. Acad. Sci. USA 89:693-697 (1992); which is incorporated herein by reference) .
  • the medium was replaced with medium containing no Polybrene .
  • the virus-containing medium was harvested and the titer determined by titrating the resulting LAPSN on D17 cells.
  • the LAPSN vector titer was determined by plating D17 target cells at 1 x 10 5 or 2 x 10 5 cells per 3.5 cm diameter well of a 6-well dish on day 1.
  • the medium was replaced with medium containing 4 ⁇ g of Polybrene per ml, and the virus stock added.
  • the cells were stained for AP+ foci as previously described. Thirty-two of the thirty-eight clones were capable of packaging LAPSN.
  • the second screen was similar to the first, except that the clones were selected in G418 after infection by LAPSN (PE501) to be certain that all cells carried the LAPSN vector.
  • the secondary screen was initialized by plating aliquots of the clones at 5xl0 4 cells per 3.5 cm diameter well of a 6 well dish on day 1. Two wells were plated for each clone. On day 2, the medium was replaced with fresh medium containing 4 ⁇ g Polybrene per ml, and 400 ⁇ l of LAPSN (PE501) was added to one well for each clone. On day 4, the medium was replaced in both wells for each clone with medium containing 0.5 mg active G418 per ml. The cells were split
  • LAPSN titers determined as in the primary screen, expressed as AP+ foci per ml of test medium, represent the mean of duplicate assays in which each value varied from the mean by no more than 17%.
  • PD clone 223 (PD223) was also found to produce the highest titer of LAPSN in both the primary and secondary screens .
  • PD223/LAPSN cells were cloned to obtain a high titer producer line of LAPSN (PD223) .
  • PD223 a high titer producer line of LAPSN
  • PA317 LAPSN
  • individual cells expressing AP on their surface were sorted into wells of a 96 well plate by FACS using an anti-AP antibody. Briefly, this assay comprised plating PD223 cells at 10 5 cells per 3.5 cm diameter well of 6 -well dishes on day 1.
  • the medium was replaced with fresh medium containing 4 ⁇ g/ml Polybrene, and either 200 ⁇ l (MOI-1) or 20 ⁇ l (MOI-0.1) of LAPSN (PA317) -containing medium was added.
  • the LAPSN (PA317) had previously been found to have a titer of lxlO 6 AP + FFU/ml on NIH 3T3 cells.
  • the cells were prepared for FACS on day 4 by treatment with trypsin, dilution in 2 ml phosphate buffered saline (PBS) containing 2% FBS and centrifugation at 1400 rpm for 5 minutes.
  • PBS ml phosphate buffered saline
  • the cell pellet was resuspended in 50 ⁇ l blocking solution from hybridoma 2.4G.2 containing 0.25 ⁇ l biotin-labeled monoclonal anti-human placental alkaline phosphatase clone 8B6 (Dako) antibody and incubated at room temperature in the dark for 30 minutes.
  • the cells were resuspended in 2 ml PBS containing 2% FBS, centrifuged, and the cell pellets were resuspended in 50 ⁇ l PBS containing 2% FBS and 0.5 ⁇ l phycoerytherin- labeled streptavidin.
  • the cells were incubated with the phycoerytherin- labeled strepavidin at room temperature in the dark for 30 minutes followed by resuspension in 2 ml PBS containing 2% FBS.
  • the cells were pelleted by centrifugation followed by resuspension in 3 ml PBS containing 2% FBS, 1 ⁇ g/ml propidium iodide.
  • the cells were sorted with a Becton Dickinson FACS Vantage Cell Sorter equipped with an automated cell disposition unit (San Jose, CA) . Dead cells and cellular debris were gated out based on forward and side scatter and propridium iodide staining.
  • fluorescence profile showed two populations: the nontransduced population, which fluoresced at an intensity equal to nontransduced control cells, and a transduced population that fluoresced at a level ten times as intense as the control cells.
  • Single transduced, fluorescent cells were sorted into individual wells of a 96 -well plate. Two wells of the plate were seeded with 500 cells each, and were later combined as the "bulk" population. Cell colonies grew in 28 of the wells. These 28 clones were split into individual wells of 24 -well plates in preparation for harvest of the medium once they were confluent . The clones matured in two phases; 21 reached confluency on day 1 and day 2 of the harvests, and the other 7 reached confluency on days 4, 5, and 6.
  • the harvested medium was used to infect D17 cells, and a primary screen was conducted by using flow cytometry essentially as described below, with an anti-AP antibody to determine what percentage of D17 cells had been infected.
  • the primary screen comprises plating D17 cells at 5xl0 4 per well of 24 well plates on day 1. On day 2, the medium was replaced with fresh medium containing 4 ⁇ g/ml • Polybrene, and exposed to 5 ⁇ l of test medium from each clone.
  • the cells were prepared for flow cytometry as described above except that the primary antibody was a mouse anti-human placental alkaline phosphatase from clone 8B6 and the secondary antibody was FITC-conjugated anti-mouse Fc (PharMingen, San Diego, CA) .
  • the primary antibody was a mouse anti-human placental alkaline phosphatase from clone 8B6 and the secondary antibody was FITC-conjugated anti-mouse Fc (PharMingen, San Diego, CA) .
  • the secondary screen the clones selected in the primary screen were plated at 2xl0 5 cells per 3.5 cm diameter well of 6 -well plates on day 1.
  • the medium was changed, and the virus-containing medium was harvested on day 3.
  • D17 cells were infected with the medium from the clones and were stained for AP + foci to determine the LAPSN titers Table 3 shows that the best clone was determined to be PD223/LAPSN clone 14.
  • the LAPSN titer produced by the "bulk" (sorted for AP expression but noncloned) PD223/LAPSN population was low in this experiment; in a repeated experiment, the LAPSN titer was found to be 6xl0 4 AP+ FFU/ml per ml.
  • the PD223/LAPSN cl4 cells consistently produce a titer of 4xl0 5 AP+ FFU/ml. Titers of up to 5 x 10 6 have been achieved in some experiments.
  • Values are expressed as the mean of those obtained in duplicate assays in which each value varied from the mean by no more than 22% for the LAPSN titers and 13% for the LNCG titers.
  • PD223/LNCG cells were also cloned to obtain a high titer producer line of LNCG(PD223) .
  • the plasmid pLNCG was constructed by inserting the sequence encoding the green fluorescent protein (GFP) into the cloning site of plasmid pLNCX (Miller and Rosman, BioTechniques 7:980-990 (1989)).
  • GFP green fluorescent protein
  • High titer producers were selected as described for the PD223 /LAPSN clone, except that an antibody was not needed for the flow cytometry because LNCG encodes green flourescent protein.
  • PD223 cells were plated at 10 5 cells per 3.5 cm diameter well of 6 -well dishes on day 1.
  • the medium was replaced with fresh medium containing 4 ⁇ g Polybrene per ml, and either 1 ml (MOI-1) or 100 ⁇ l (MOI-0.1) of LNCG (PT67) -containing medium was added.
  • the LNCG(PT67) stock had previously been found to have a titer of 2xl0 5 GFP + FFU/ml .
  • the cells were prepared for FACS.
  • the cells were trypsinized, diluted in 2 ml phosphate buffered saline (PBS) containing 2% FBS, centrifuged at 1400 rpm for 5 min, and resuspended in 3 ml PBS containing 2% FBS and 1 ⁇ g/ml propidium iodide.
  • the vector expresses Green Fluorescent Protein which provides a visible signal when excited by blue light of the appropriate wave length. Propidium iodide also added in this assay.
  • the cells were sorted with a Becton Dickinson FACS VANTAGE Cell sorter equipped with an automated cell deposition unit (Becton Dickinson, San Jose, CA) .
  • the cells were gated as described above and single GFP-positive cells sorted into individual wells of 96-well plates. Two bulk populations were also sorted, and later combined to yield the "bulk" population. Cells expressing GFP were sorted into wells of a 96-well plate and colonies were split into wells of a 24 -well dish as they matured. For a primary screen, medium samples from 30 clones were harvested and used to infect D17 cells, and the percent infected was analyzed by flow cytometry. Five clones were selected for a secondary screen.
  • clones selected in the primary screen were plated at 2xl0 5 cells per 3.5 cm diameter well of 6 -well plates on day 1. On day 2, the medium was changed, and the virus-containing medium was harvested on day 3. D17 cells were infected with the medium from the clones and were observed under blue light to determine the LNCG titers. Table 3 shows that all selected clones packaged LNCG at a similar rate that was higher than that by the "bulk" (sorted for positive GFP expression but noncloned) population, and that two clones, clone 14 and clone 24, packaged LNCG at the highest rate.
  • Viral interference experiments were conducted to determine whether a vector packaged by the PD223 cells uses the same receptor as that used by MDEV (Table 4) .
  • Viral interference experiments rely on the observation that a cell that is producing a retroviral Env is resistant to infection by a retrovirus that uses the same receptor as the expressed Env.
  • the entry of LAPSN (PD223) was dramatically impeded by the presence of MDEV that had been activated by hydrocortisone (>2xl0 4 fold inhibition), while the entry of LAPSN(PA317) was equivalent on both cell types.
  • LAPSN(PD223) and LAPSN(PA317) contain the same Gag and Pol proteins, indicating that the inhibition was Env-specific and at the level of viral entry.
  • LAPSN LAPSN Titer FFU/ml on pseudotype dunni/N2 dunni/N2+MDEV Fold Interference PD223 6xl0 4 ⁇ 2.5 >2xl0 4
  • the titer is expressed as the mean of duplicate assays in which each value varied by no more than 6% of the mean. The experiment was repeated with nearly identical results.
  • the LAPSN (PD223) stocks had a titer of 3xl0 5 AP + FFU/ml on D17 cells.
  • PA317 refers to a packaging cell line that pseudotypes vectors in the amphotropic MLV envelope.
  • a preferred packaging cell line is capable of packaging retroviral vectors without producing contaminating replication-competent retroviruses (RCR) , or helper virus.
  • S + L " assays are often used to detect helper virus, but these were not used here because previous experiments have shown that wild-type MDEV does not score on S + L " assays utilizing PG4 cells, CCC-81 cells overlaid with NRK cells, MvlLu cells, or SC-1 cells overlaid with MvlLu cells (Miller et al . , J. Virol. 70:1804-1809 (1996)).
  • test medium harvested from PD223/LAPSN cells was placed on G355/LAPSN or dunni/LAPSN cells.
  • the G355/LAPSN and dunni/LAPSN cells were passaged for more than two weeks, and then the medium was transferred to G355 or M. dunni cells.
  • Test medium which contains RCR capable of replication on the cells package the LAPSN vector, allowing its detection on the final indicator cells.
  • the ⁇ same type of cells were used for the final indicator cells as the LAPSN-containing amplification cells to ensure that any amplified virus could infect the indicator cells.
  • Two assays using different cell types were used to enhance the probability of detecting RCR.
  • the packaging cell medium was tested for RCR by first plating G355/LAPSN or dunni/LAPSN cells at 2xl0 5 cells per 6 cm diameter dish in 3 ml of medium on day 1. On day 2, the test stock (packaging cell medium) was added in the presence of fresh medium containing 4 ⁇ g/ml of Polybrene . The cells were passaged in the presence of Polybrene to facilitate viral spread, during which time they were split 1:10 every several days. On day 17, the medium was harvested and titered on G355 cells for the G355 assay or M. dunni cells for the dunni assay. In all cases, the dunni cells had not been exposed to agents that activate the endogenous MDEV and so were not expressing MDEV.
  • the LAPSN titer was determined on the final indicator cells as described previously.
  • the titer (AP + FFU/ml) was expressed as the mean of those determined by duplicate assays in which each value varied from the mean by no more than 20% of the mean for the G355 assay and 4% of the mean for the dunni assay.
  • Table 5 shows that both the G355 and dunni assays were able to detect wild-type MDEV, and both assays can also efficiently detect amphotropic MLV. Neither assay, however, detected any RCR in the medium harvested from PD223/LAPSN cells. TABLE 5
  • CHO cells are known to be difficult to transduce with retroviral vectors without pretreatment with tunicamycin, with a block at the level of viral entry (Miller and Miller, J. Virol. 65:78-84 (1992)).
  • CHO cells were plated at lxlO 5 cells per 3.5 cm diameter well of a 6-well dish on day 1, infected with the appropriate vector on day 2, and stained for AP + foci on day 4, as described above.
  • Titers were expressed as the mean of those determined by duplicate assays, in which each value varied from the mean by no more than 18%.
  • the titers of LAPSN packaged by the various cell lines were generally low on CHO cells, as predicted. However, the titer on permissive cells (D17 or NIH 3T3) was high for each stock, demonstrating that there was functional virus present that did not transduce the CHO cells. The most efficient entry into CHO cells was achieved by LAPSN (PD223) .
  • This example describes at least six different receptors used by murine retroviruses for entry into M. dunni cells.
  • Pseudotypes of a retroviral vector encoding human placental alkaline phosphatase (AP) was used to determine interference in M. dunni cells.
  • AP placental alkaline phosphatase
  • the following replication-competent retroviruses were used in interference assays described in more detail below: 1387, MoMLV strain 1387; AKR623, AKR MLV strain 623; IE, Friend MCF strain IE; 4070A and 1504A, amphotropic MLV strains 4070A and 1504A (Chesebro and Wehrly Virol . 141:119- 129 (1985)) .
  • the MLV-K strain of MoMLV was obtained from NIH 3T3 cells transfected with the pMLV-K clone of MoMLV (Miller and Verma, J. Virol. 49:214-222 (1984)).
  • NZB xenotropic virus was obtained from M.
  • dunni cells transfected with the circularly permuted NZB molecular clone NZB 9-1 (O'Neill et al., J. Virol. 53:100-106 (1985)) after cutting the plasmid with Eco RI and religating the DNA to generate intact NZB provirus circles.
  • 10A1 virus was obtained from NIH 3T3 cells transfected with the permuted 10A1 virus DNA clone pB6 (Ott et al., J. Virol. 64:757-765 (1990)) after cutting the plasmid with Sal I and religating the DNA to generate intact 10A1 provirus circles.
  • the LN retroviral vector encodes neomycin phosphotransferase (Miller and Rosman, BioTechni ⁇ ues 7:980-990 (1989) )
  • the LAPSN vector encodes neomycin phosphotransferase and human placental alkaline phosphatase (AP) (Miller et al . , Proc. Natl. Acad. Sci. USA 91:78-82 (1994) ) .
  • M. dunni tail fibroblasts (dunni cells) (Lander and Chattopadhyay, J. Virol. 52:695-698 (1984)) were grown in Dulbecco's modified Eagle's medium with 4.5 g/1 glucose and 5% fetal bovine serum at 37°C in a 10% C0 2 /air atmosphere.
  • PE501 ecotropic, PM571 polytropic, and PA317 amphotropic retrovirus packaging cell lines were grown under similar conditions except that 10% fetal bovine serum was used.
  • dunni/LN and dunni/LAPSN cells were made by transduction with helper- free vector stocks produced by PA317 retrovirus packaging cells containing the vectors .
  • dunni/LN and dunni/LAPSN cells were infected with replication-competent viruses by seeding the cells the day before infection at 10 5 per 6 cm dish, infecting the cells with 100 ⁇ l virus stock in the presence of 4 ⁇ g/ml Polybrene (Sigma) , and passaging the cells for ⁇ ll days in the absence of Polybrene to allow complete virus spread.
  • Virus was harvested from confluent layers of cells 16 h after a medium change and frozen at -70°C.
  • Helper-free retroviral vectors were generated from retrovirus packaging cells as previously described (Miller and Rosman, BioTechniques 7:980-990 (1989); incorporated herein by reference in its entirety) .
  • the envelope protein in PE501 cells is from MoMLV molecular clone pMLV-K, that in PM571 is from Friend MCF strain 98D, and that in PA317 cells is from amphotropic MLV strain 4070A. Chesebro and Wehrly (Virol . 141:119-129 (1985); incorporated herein by reference) previously assigned viruses to different interference groups which exhibited interference in M. dunni cells.
  • ecotropic virus 1387 but not ecotropic virus AKR623, interfered with polytropic viruses 98D and 1512, and both ecotropic viruses interfered with infection by xenotropic virus AKR6.
  • Amphotropic virus 4070A but not amphotropic virus 1504A, interfered with xenotropic virus AKR6 and ecotropic virus 1387 and with xenotropic virus AKR6.
  • Polytropic virus IE interfered with ecotropic virus 1387. Challenge by amphotropic viruses was not analyzed because amphotropic virus-specific antibodies were not available for virus detection in the immunofluorescence assay used.
  • the M. dunni tail fibroblasts used previously (Chesebro and Wehrly, supra . ) , were obtained and transduced with the LN retroviral vector (Miller and Rosman, supra.), that expresses neomycin phosphotransferase, or with the LAPSN vector (Miller et al . , supra . 1994), that expresses neomycin phosphotransferase and human placental alkaline phosphatase (AP) , by using helper virus-free vector stocks made from PA317 retrovirus packaging cells (Miller and Buttimore, Mol. Cell. Biol. 6:2895-2902 (1986)) containing the vectors.
  • dunni/LN and dunni/LAPSN cells did not produce the vectors in the absence of added replication-competent virus.
  • the original retroviruses used previously (Chesebro and Wehrly, supra.) were obtained and used to infect the dunni/LN and dunni/LAPSN cells.
  • the presence of replication-competent retrovirus was confirmed in all cases by measurement of the production of the LN or LAPSN vectors. Interference assays were performed by first seeding infected (dunni/LN cells infected with the interfering virus) and uninfected dunni/LN cells at 5 x 10 4 per 3 cm-diameter well of 6-well culture trays on day one.
  • the medium was replaced with 2 ml medium containing 4 ⁇ g/ml Polybrene and the challenge pseudotyped LAPSN vectors.
  • the cells were stained for alkaline phosphatase-positive foci as described (Fields- Berry et al., Proc. Natl. Acad. Sci. USA 89:693-697 (1992). Transduction (gene transfer and expression) by the LAPSN vector was measured by staining for foci of alkaline phosphatase-positive cells.
  • Results were expressed as the log 10 of the fold interference, or log 10 [(LAPSN vector titer on uninfected dunni/LN cells) / (LAPSN vector titer on dunni /LN cells infected with the interfering virus) ] .
  • the presence of the LN vector in the target cells did not interfere with transduction by the LAPSN vector or with detection of AP expression, and was only included to allow detection of the presence of the interfering viruses by measurement of LN vector production.
  • the LAPSN vector with the indicated pseudotypes was used to infect dunni/LN cells or dunni/LN cells infected with the indicated interfering MLVs, and the apparent vector titer in FFU/ml were determined.
  • the vector titer is expressed in the Table as the log 10 of the mean titer determined on dunni/LN cells, and is an arithmetic mean of at least two experiments.
  • Interference values are expressed as the log 10 of the fold interference, or log 10 (LAPSN titer on uninfected dunni/LN cells) / (LAPSN titer on dunni/LN cells infected with the interfering virus) .
  • the fold interference was calculated as the arithmetic mean of at least two independent determinations, which varied by no more than 58% from the mean. Boxed areas indicate interference between viruses in the same interference group. Not determined.
  • LAPSN virus produced by PE501 ecotropic packaging cells behaved like the ecotropic LAPSN (1387) virus.
  • PE501 cells were constructed by using the pMLV-K clone of MoMLV (Miller and Verma, J. Virol. 49:214-222 (1984)), and 1387 virus represents a different isolate of MoMLV.
  • LAPSN produced by PM571 Friend MCF strain 98D-based packaging cells (Miller and Miller, J. Virol.
  • LAPSN pseudotyped with the xenotropic NZB virus behaved like the xenotropic AKR6 pseudotype.
  • LAPSN (PA317) behaved like the other amphotropic pseudotypes of the vector, but the best correlation was with LAPSN (4070A) , which was expected since PA317 cells were constructed by using env from a molecular clone of 4070A.
  • the results obtained when the interfering virus was 1387 were somewhat anomalous.
  • the 1387 virus-infected dunni/LN cells grew slowly, displayed many multinucleated cells, and exhibited high rates of cell death, a phenomenon that persisted even with prolonged cultivation.
  • Infection rates observed for non-ecotropic LAPSN pseudotypes in 1387 virus-infected dunni/LN cells were low compared to those obtained in uninfected dunni/LN cells is most likely due to cell death and slow growth of the 1387 virus-infected dunni/LN cells rather than interference by the 1387 virus.
  • the continued presence of multinucleated cells in the dunni/LN cells infected with 1387 virus indicates that the receptor used by 1387 virus is not effectively blocked by the virus, resulting in continued virus-mediated cell fusion, and provides an explanation for the poor interference of the 1387 virus with ecotropic pseudotype LAPSN viruses .
  • M. dunni cells were infected in parallel with 1387 virus harvested from 1387 virus-infected dunni/LAPSN cells, or with virus harvested from NIH 3T3 cells transfected with a DNA clone of MoMLV, pMLV-K (Miller and Verma, supra.), and the cells were observed during several passages in culture. The presence of large multinucleated cells that increased in number with time was observed in M.
  • dunni cells infected with either virus preparation although it was most pronounced in cells infected with 1387 virus.
  • M. dunni cells were relatively efficiently infected by the LAPSN vector with a 1387 (MoMLV) pseudotype, the apparent titer on M. dunni cells being 10 6 FFU/ml (Table 7) .
  • MoMLV molecular multi-layer vector
  • Table 7 M. dunni tail fibroblasts in common use, and the use of different cells by different labs might explain the these results.
  • the type used here can be recognized by the unusual characteristic that confluent layers of the cells secrete molecules that render the culture medium highly viscous, a property that is not shared with the M. dunni cells used in other studies
  • dunni-v and dunni- nv to denote viscous and non-viscous medium, respectively.
  • Dunni-nv cells (Eiden et al . (1993), supra.) were directly compared with the dunni-v cells (Table 8) .
  • the apparent titer of the PE501 (MoMLV) pseudotype LAPSN vector was similar in both dunni cell strains, but was up to 250-fold lower in dunni cells as compared with NIH 3T3 cells.
  • the titer of the 1387 (MoMLV) pseudotype LAPSN vector was also similar on both dunni cell strains, but was reduced by only about 6-fold in dunni cells compared with NIH 3T3 cells.
  • the titer of amphotropic LAPSN vector was similar in both M. dunni cell strains and in NIH 3T3 cells.
  • the ecotropic virus AKR623, which is not an MoMLV strain, promoted LAPSN infection of dunni-nv cells at the same rate as NIH 3T3 mouse cells, but at a 20-fold lower rate in dunni-v cells.
  • virus strain- and cell strain-dependent variations in ecotropic-pseudotype vector infection of M While there are virus strain- and cell strain-dependent variations in ecotropic-pseudotype vector infection of M.
  • dunni cells both M. dunni cell lines can be infected by virions carrying the MoMLV envelope.
  • the markedly lower rate of infection in M. dunni cells in comparison to NIH 3T3 cells seen for one strain of MoMLV is consistent with prior observations (Eiden et al . , supra . (1993); Eiden et al . , supra . (1994); Lander and Chattopadhyay, supra . (1984)).
  • dunni-v cells are most likely derived from M. dunni animals, and are not cells from another species due to culture contamination or mistaken identification. This conclusion depends on Southern analyses of restriction enzyme- digested DNA samples from these M. dunni cells and from M. dunni mice, which show identical patterns of bands that hybridize to a probe made from the relatively unique endogenous virus MDEV, while DNA samples from cell lines ' derived from laboratory mice show a different pattern of weakly-hybridizing bands, and DNA samples from cells of other species show no hybridization (Example I) .
  • M. dunni (dunni-v) cells (Table 9) .
  • Ecotropic and MDEV viruses showed no interference with any of the other groups.
  • 10A1 and amphotropic viruses showed nonreciprocal interference, but no interference with any of the other groups.
  • polytropic and xenotropic groups showed nonreciprocal interference, but no interference with any other groups.
  • the reason for the more complicated interference patterns previously observed among these groups in M. dunni cells is likely due to the assay employed, which required spread of the challenge virus after infection to produce a signal, and virus spread might have been inhibited by the interfering virus.
  • murine retroviruses can be divided into six interference groups that use at least six different receptors for entry into M. dunni cells.
  • the LAPSN vector with the indicated pseudotypes was used to infect dunni/LN cells or dunni/LN cells infected with the indicated interfering MLVs, and the apparent vector titer in FFU/ml were determined.
  • the vector titer is indicated in the Table as the log 10 of the mean titer determined on dunni/LN cells, and is an arithmetic mean of at least two experiments. Interference values are expressed as the log 10 of the fold interference, or log, 0 (LAPSN titer on uninfected dunni/LN cells) / (LAPSN titer on dunni/LN cells infected with the interfering virus) .
  • the fold interference was calculated as the arithmetic mean of at least two independent determinations, which varied by no more than 58% from the mean.
  • MDEV was activated from the cells of the Asian wild mouse during testing of human cells for replication-competent virus in human gene transfer trials (Miller et al . J. Virol. 70:1804-1809 (1996)). MDEV did not appear to match the descriptions of previously known viruses and was shown to use a unique receptor and does not belong to any of the murine retrovirus interference groups (Example IV) . This Example describes tests for interference between MDEV and viruses from additional interference groups.
  • M. dunni tail fibroblasts (Chattopadhyay et al . , Virology 113:465-483 (1981)), PA317 amphotropic retrovirus packaging cells, PG13 gibbon ape leukemia virus (GALV) -based packaging cells, NIH 3T3 thymidine kinase-negative cells (Miller et al . , Mol. Cell. Biol.
  • C2C12 cells ATCC CRL 1772
  • HeLa cells ATCC CCL 2
  • D17 cells ATCC CCL 183
  • CCC- 81 cat cells transformed with Moloney murine sarcoma virus Fischinger et al . , Proc. Natl. Acad. Sci. USA 72:5150-5155 (1975)
  • 5637 human bladder carcinoma cells ATCC HTB 9
  • IB3 cells Zeitlin et al . , Am. J. Respir. Cell Mol. Biol.
  • HT1080 cells ATCC CCL 121
  • NRK cells DeLarco, et al., J. Cell Physiol . 94:335-342 (1978)
  • DMEM Dulbecco's modified Eagle's medium
  • FBS fetal bovine serum
  • HDF primary human dermal fibroblasts
  • G355 feline embryonic glial cells and G355 cells infected with RD114 endogenous cat virus were grown as described in Example 1.
  • CHO-K1 cells (ATCC CCL 61) were grown in ⁇ MEM with 10% FBS.
  • HUT 78 cells (ATCC TIB 161) were grown in RPMI 1640 plus 10% FBS.
  • QT35 cells (Moscovici et al . , Cell 11:95-103 (1977)) were grown in Ham's F10 medium with 10% tryptose phosphate broth, 1% chick serum, 1% DMSO, 5% FBS, and 2% sodium bicarbonate (7.5% solution) .
  • LAPSN infection was scored by histochemical staining for AP+ foci of cells two days after infection as described (Miller and Chen., J. Virol. 70:5564-5571 (1996); incorporated herein by reference in its entirety) .
  • the replication competent viruses used in this Example included ecotropic Moloney murine leukemia virus (MoMLV) (pMLV-K; Miller and Verma, (1984) supra.), amphotropic virus (AM-MLV; Miller et al . , Mol. Cell. Biol. 6:2895-2902 (1986)), NZB xenotropic (O'Neill et al . , J. Virol. 53:100-106 (1985) ) , polytropic (MCF) virus strain 98D (Chesebro and Wehrly, (1985) supra.), 10A1 (Ott et al .
  • MoMLV Moloney murine leukemia virus
  • AM-MLV amphotropic virus
  • AM-MLV Miller et al .
  • Mol. Cell. Biol. 6:2895-2902 (1986) NZB xenotropic
  • MCF polytropic virus strain 98D
  • 10A1 Ott et al .
  • MMTV mouse mammary tumor virus
  • SNV-pseudotype LAPSN vector was made by infecting D17/SNV cells with helper- free amphotropic LAPSN vector produced from PA317 cells, selecting the cells in G418, growing the G418-resistant cells in the absence of G418, and harvesting virus from confluent dishes of cells about 16 h after a medium change.
  • RD114 -pseudotype LAPSN vector was made in a similar manner following infection of G355/RD114 cells with the helper- free LAPSN vector.
  • MDEV is not in the GALV or RD114 retrovirus interference groups, and MDEV uses a different receptor for entry than those used by other murine leukemia viruses by interference analysis (Table 9, Miller et al . , (1996) supra.).
  • Tests for interference between MDEV and viruses from additional groups including the gibbon ape leukemia virus (GALV) and the RD114 cat endogenous virus groups were carried out. These viruses were chosen because members of the GALV interference group have been found to interfere with an endogenous retrovirus from the Asian mouse M. caroli (Callahan et al . , p.689-713. In H. C. Morse III (ed.), Workshop on the origins of inbred mice, Academic Press, Inc., New York (1978); Lieber et al . , Proc. Natl. Acad. Sci. USA 72:2315-2319
  • a Uninfected HDF cells and HDF cells infected with the indicated interfering viruses were exposed to LAPSN vectors with the indicated pseudotypes and were stained for AP+ foci of cells two days after infection. Values are means of duplicate assays in a single experiment which varied by no more than 33% from the mean (except for the value 1, which represents the mean of 0 and 2 FFU/ml) . The experiment was repeated with similar results.
  • b PG13 refers to a packaging cell line which produces GALV- pseudotype virus.
  • c PA317 refers to a packaging cell line which produces amphotropic-pseudotype virus.
  • Table 11 shows the results of additional interference assays performed in D17 canine cells using additional representatives of the RD114 interference group.
  • RD114, SNV and MPMV do not Interfere with Transduction by a
  • MDEV has a wide host range, and MDEV-pseudotyped LAPSN virus was able to infect a variety of cell types from all species tested, including mouse, rat, hamster, quail, cat, dog and human cells (Table 13) .
  • the titer of LAPS (MDEV) was between 10 5 and 5 x 10 ⁇ on all cells tested, except for Balb/c 3T3 cells, for which the titer was only 200 FFU/ml. No phenotypic changes were observed in cells infected with MDEV except in experiments with the HUT 78 human T cell line, in which an increase in doubling time and very large multinucleated cells were observed.
  • the ability of MDEV- pseudotype vector to infect different cell types from multiple species indicates that the receptor used by this virus is widely distributed. TABLE 13 Host Range of MDEV a
  • the indicated cells were exposed to MDEV-pseudotype LAPSN and were stained for AP+ foci of cells two days after infection. Values are means of duplicate assays in a single experiment which varied by no more than 25% from the mean. The experiment was repeated with similar results .
  • the LAPSN (MDEV) titer on HeLa cells was measured by production of G418 -resistant colonies (CFU/ml) rather than AP+ foci because HeLa cells have very high levels of endogenous alkaline phosphatase.
  • a virus produced from laboratory mouse-derived cell lines by activation with IdU is distinct from MDEV.
  • NIH 3T3, C2C12, and Balb/c 3T3 laboratory mouse cells were exposed to either IdU or hydrocortisone.
  • Balb/c 3T3, NIH 3T3 , or C2C12 cells were seeded in 6 -well plates at 4 xl0 ⁇ cells per 3.5 cm-diameter well on day one.
  • duplicate wells were treated with culture medium saturated with 5-iodo-2'deoxyuridine (IdU), medium containing 90 ⁇ M hydrocortisone 21-phosphate (Sigma) , or medium alone for 24 h.
  • IdU 5-iodo-2'deoxyuridine
  • the cells were trypsinized and co-cultivated with D17/LAPSN cells to allow for replication of viruses that might be unable to replicate in the mouse cells, and to allow detection of replication-competent retroviruses by measurement of LAPSN vector production.
  • medium exposed to the cell mixtures for 24 h was collected, filtered (0.45 ⁇ m pore size), and tested for the presence of LAPSN on M. dunni cells.
  • the exposed cells were then cocultivated with D17/LAPSN cells to allow for replication of viruses that might be unable to replicate in the mouse cells, and to allow detection of replication-competent retroviruses by measurement of LAPSN vector production.
  • Balb/c 3T3 IdU-induced retrovirus BIRV
  • Duplicate wells of Balb/c 3T3 cells which were treated with hydrocortisone or were untreated remained negative for the production of virus for the seven week duration of the experiment.
  • replication- competent virus could not be detected after identical treatment of either NIH 3T3 or C2C12 cells.
  • dunni cells in that xenotropic virus strongly inhibits polytropic vector transduction (10 5 -fold) , while polytropic virus much less severely inhibits transduction by xenotropic virus ( ⁇ 40-fold) .
  • BIRV is capable of infecting M. dunni , NIH 3T3, G355, and primary human fibroblasts (Table 15), indicating that BIRV is a polytropic virus, since xenotropic viruses do not infect cells from laboratory mice.
  • MDEV 3 6xl0 4 ND b ND Polytropic 4xl0 5 400 ⁇ 1 lxlO 5 Xenotropic 4x10 s ⁇ 2 1 6 Amphotropic 4xl0 5 5xl0 4 ND ND
  • the indicated cells were infected with BIRV-pseudotype LAPSN and were stained for AP+ foci of cells two days after infection. Shown here are the titers of a population of cells producing LAPSN (BIRV) . Similar results were obtained using a second, independently IdU- induced bulk population of cells producing BIRV.
  • MDEV shares a basic proviral structure with other retroviruses (LTR-gag-pol-env-LTR) , but this virus is distinctly different from other retroviruses in several respects.
  • the first unusual feature of MDEV is that it represents a novel murine viral interference group distinct from the amphotropic, xenotropic, ecotropic, polytropic, and 10A1 groups.
  • MDEV does not interfere with SNV, MPMV, or GALV.
  • MDEV appears to share a receptor with the RD114 virus in G355 cat cells, it does not do so in the other target cells tested.
  • BIRV endogenous virus
  • MDEV is unusual with regard to its exceptionally large host range. MDEV is able to efficiently infect every cell type tested with the exception that Balb/c 3T3 cells were infected at a low level. Discrepant results in CCC-81 cells may relate to the variable interference of RD114 with MDEV infection, since cat cells carry and potentially could express the endogenous RD114 virus . Finally, MDEV appears by hybridization analysis to have an unusual genome. Previous studies were not able to detect endogenous virus in M.
  • dunni cells using either a reverse-transcriptase-polymerase chain reaction with primers to various parts of the env gene, which could detect xenotropic, ecotropic, polytropic and modified polytropic viruses (Irving et al . , Biotechnology 11:1042-1046 (1993)), or by hybridization using probes for several MLV DNAs (Lander et al., J. Virol. 52:695-698 (1984)). As shown herein, MDEV does not hybridize to the genomes of any of the other viruses tested, including amphotropic, ecotropic, xenotropic, polytropic, 10A1, GALV, RD114, and MMTV.
  • MDEV arose from an endogenous provirus present in M. dunni mice. MDEV appears to use a different receptor for cell entry than those of other retroviruses. The ability of MDEV to efficiently infect many types of human cells indicates that retrovirus packaging cells based on MDEV can be used for gene transfer purposes and human gene therapy applications.
  • the packaging cell line PD 227 was deposited with the American Type Culture Collection, 10801 University Boulevard, Manassas, Virginia 20110-2209, , 1998, under the conditions of the Budapest Treaty and designated accesion number .
  • CTAAACTCCT TAACATTCCT GAACTCTTCT TCACCCCAGA GTCCAACCCC TCCCATCTAG 8040
  • CTAAACTCCT TAACATTCCT GAACTCTTCT TCACCCCAGA GTCCAACCCC TCCCATCTAG 8280
  • CAAGATGTTA CACAGCCCCC TTAATTACGC AGAACTCCCC TGGCAGAACA CCTTGACCTT 8460
  • Gly Ser Gly Cys Gly lie Pro Pro Arg Lys Glu Gly Ser Gly Cys Gly 35 40 " 45 lie Pro Pro Arg Gin Glu Gly Ser Arg Cys Gly He Arg Pro His Pro 50 55 60
  • Trp Arg Asp Pro Glu Gly Gly Gin Thr Gly Gin Leu Thr Trp Thr Arg 290 295 300

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Abstract

L'invention concerne des cellules d'encapsidation rétrovirales produisant des particules rétrovirales à réplication défective, capable de se fixer à des récepteurs rétroviraux du virus endogène Mus dunni sur des cellules cibles. Ces cellules d'encapsidation, qui sont utiles au transfert génique et en thérapie génique, utilisent un vecteur codant une protéine Env rétrovirale M. dunni, et produisent lesdites particules rétrovirales à titrage élevé.
PCT/US1998/009452 1997-05-09 1998-05-08 LIGNEES CELLULAIRES D'ENCAPSIDATION RETROVIRALES ENDOGENES DE $i(MUS DUNNI) WO1998050538A1 (fr)

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